My apologies if in my ignorance I committed a faux pas in sending Tracey
Wheeler's Golgi Cox protocol as an attachment--trying again here with plain
text in hopes that this helps (ps Tracey can be reached at twheele2@gmu.edu
if you have questions):
Golgi Cox Protocol
(Tracey's way.there are others as well, so don't be nervous if you come
across them!)
Step 1: Prepare the Golgi-Cox solution
Solution A: 5% Potassium Dichromate in distilled H2O
200ml distilled H2O + 10 grams Potassium Dichromate
(Mix in a glass beaker using a glass rod - best to do under fume hood)
Solution B: 5% Mercuric Chloride (sublimate) in distilled H2O
200ml distilled H2O + 10 grams Mercuric Chloride
(Mix in a glass beaker using a glass rod)
(Mix solution on top of hotplate (on 5), stirring until dissolved)
(Must be done under fume hood)
Solution C: 5% Solution of Potassium Chromate in distilled H2O
160ml distilled H2O + 8 grams Potassium Chromate
(Mix in a glass beaker using a glass rod - best to do under fume hood)
Mix Solution A and Solution B into a 500ml glass beaker.
Mix Solution C and 400ml of distilled H2O into a 1,000ml + glass beaker.
Slowly pour the AB Solution into the C Solution while stirring continuously
with a glass rod.
Store in a glass stoppered bottle for 5 days in the dark.
Note: You can easily manipulate the quantity of solution. Just make sure to
follow these ratio's
5 Volume parts of 5% Potassium Dichromate solution
5 Volume parts of 5% Mercuric Chloride solution
4 Volume parts of 5% Potassium Chromate solution
10 Volume parts of H20 (to add to PC solution)
Step 2: Transfer Golgi-Cox solution into small glass bottles.
Use a plastic syringe to remove the GC solution from the large glass
bottle(s).
Be sure to avoid the reddish precipitate that formed on the top and bottom
of
the bottle.
Glass bottles should be filled about ¾ full (to save room for 1 rat brain).
Step 3: Sacrifice Rats using Saline Perfusion Technique.
Deeply anaesthetize the animal.
Place on an empty breeder box with wire top. (to allow blood to drain into
box)
Open chest cavity to expose heart.
Insert 60 ml syringe filled with 9% saline into bottom right chamber of the
heart. (This would be the animal's left chamber)
Using scissors, cut the bottom left chamber of the heart open. (This would
be the animal's right chamber)
Begin to slowly push saline through the animal's system until the blood
leaving the left chamber is clear. (This may take 3 syringes of saline.)
When fluids are clear, decapitate and remove brain.
Drop whole brain into prepared bottle(s) of Golgi Cox solution.
Place in the dark for 14 days, refresh solution after 2 days.
Step 4: Transfer Brains into Sucrose Solution.
Mix 300 grams of Sucrose into 1000ml of distilled H2O.
Place Beaker over hotplate and stir (using stir bar) until dissolved.
Cool in refrigerator. (Once cool, ready to use.)
Empty GC solution from jar and place brain on chem. wipe paper.
Slightly blot.
Rinse jar in distilled H2O, and refill with Sucrose Solution ¾ full. (In
order to save room for brain.)
Place brain in jar with Sucrose Solution. Brain will Float.
Place jar(s) in refrigerator.
(Once brains sink, they are ready to be sectioned.)
(Brains should be sectioned within 2 months of transfer into sucrose.)
Step 5: Section using a Vibratome.
Prepare razor blade by immersion in xylene for 5 minutes to remove any
traces of
oil. (This should be done under the fume hood.) Wipe blade dry.
Prepare a 6% sucrose solution. (6 grams sucrose in 100ml distilled H2O.)
Mix well and make sure it is at room temperature or below before using.
Fill the vibratome reservoir with the 6% Sucrose solution until the blade is
covered.
Mount brain section (up to ½ a full brain) onto vibratome platform using
superglue. (Make sure tissue is adhered well before sectioning, 5-7 minutes
or more.)
Insert platform (with adhered brain section) into reservoir.
Set the vibratome speed and amplitude around the midpoint for sectioning
(adjust as necessary for your specific machine and comfort level.)
Section at 200 micro meters or desired thickness. (Sections over 400 may be
difficult to analyze.)
Using a small paintbrush coax the section onto a gelatinized slide.
Cover tissue with Parafilm. Place slide on flat surface covered with
bibulous
paper. Place another sheet of bibulous paper over paraflim. Place your
palm over the section an press down slightly, being careful not alter
movement. (Your goal is to press the section into the gelatin on the slide
so it
will adhere to slide during staining.)
Remove bibulous paper and place in humidity chamber.
Note: We used a water sleeved incubator. In this apparatus it is necessary
to
keep the parafilm on the section. We also placed the slides on plastic trays
which also held capfuls of water to be sure the slides did not dry out.
Do not keep the slides in storage (humidity chamber or water incubator) for
more than 4 days.)
Step 6: Staining
Prepare fresh solutions (enough to cover all slides):
Distilled H2O (3 total)
Ammonium Hydroxide (1 total - keep under fume hood!)
Kodak Fix (1 total - keep under fume hood, mix in dark)
50% alcohol (1 total)
70% alcohol (1 total)
95% alcohol (1 total)
100% alcohol (3 total)
CXA solution (1 total - keep under fume hood!)
Kodak Fix Solution:
Prepare all ingredients in beakers
Mix in order: (do not mix in light)
1010 ml distilled H2O (add)
251 ml Kodak Fix solution A (add)
28 ml Kodak Fix solution B (add)
2020 ml distilled H2O
CXA Solution:
1000 ml Chloroform
1000 ml Xylene
1000 ml 100% Alcohol
(you can manipulate amounts, just use 1/3 each)
Remove parafilm from slides if necessary and place in slide
rack. Dip according to process below:
1. Distilled H2O for 1 minute
2. Ammonium Hydroxide for 30 minutes (IN THE DARK)
(Using a darkroom light mix the Kodak Fix solution now.)
3. Distilled H2O for 1 minute (Best to just keep lights off)
4. Kodak Fix solution for 30 minutes (IN THE DARK)
5. Distilled H2O for 1 minute (once in H2O you can turn on
lights)
6. 50% alcohol 1 minute
7. 70% alcohol 1 minute
8. 95% alcohol 1 minute
9. 100% alcohol 5 minutes
10.100% alcohol 5 minutes
11.100% alcohol 5 minutes
12.CXA 15 minutes (Keep under fume hood)
(Keep slides in CXA under fume hood while cover slipping,
pull one slide out at a time.) Note: change gloves often - they
will disintegrate in CXA.
Step 7: Coverslip with permount and lie out to dry.
If possible all cover slipping should be done under fume hood. Slides should
be allowed to remain under fume hood for 24 hours - lying flat.
Pull one slide out of CXA at a time.
Using a glass dropper, place 2 drops of permount on top of tissue.
(Sections dry quickly, do not remove slide from CXA and allow to sit in air
for more than 20 seconds.)
Place glass coverslip over section, being careful to avoid trapping air
bubbles. Note: Too little permount could allow tissue to dry out, too much
will cause coverslip to slide off - monitor your work for these problems.
Place slide on absorbent paper (we use white tray liners - usually under rat
cages)
Allow slides to lie flat for 24 hours.
Slides can now be moved into slide boxes for storage. KEEP BOXES
OPEN!!! Slides need to continue to dry for 6 months before analysis should
be attempted.
----- Original Message -----
From: "Herb Hagler"
To:
Sent: Thursday, November 20, 2003 10:33 AM
Subject: [Histonet] Regarding HTML encoded email and unwanted attachments,
An Update
> It would be so much simpler if everyone that is using their email
> client would simply learn how to send plain text email messages. That
> simple step would eliminate a lot of the trash code that appears in
> everyone's mail messages and in the digests. All mail clients do have
> this as an option, consult your local geek if you can't find it.
>
> In the meantime I have checked with our group on campus who are
> providing the list server space. It turns out that they are presently
> running an older version of the list software that does not currently
> block attachments and does not successfully remove the html garbage
> code.
>
> They are planning to update the list server software by mid December so
> please do not send attachments in your postings to the list. This
> includes the option to attach your vcard identity.
>
> After the update we will be able to eliminate the attachments and the
> html junk that comes with some people not using plain text message
> encoding.
>
> Thanks for your help and understanding. Have a great weekend....
>
> Herb Hagler
>
>
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet